Late stage erythroblast development is emerging as a sharply regulated process. New insight into key regulators has been won via aggressive genetic and cell biology approaches as recently applied to primary erythroid cell systems (with attention paid to the step-wise nature of erythroblast formation). Examples include Bcl11a and TR2/TR4 repressors of beta-globins (1, 2); miR-150, miR-144/451 as erythro-megakaryocytic transcript decay factors (3-5); and EMP plus ICAM4 as erythroblastic island bridging factors (6, 7). In ongoing studies of murine bone marrow (BM) erythropoiesis, the PI has observed a striking in vivo expansion capacity for a selective cohort of late-stage BM erythroblasts, specifically a KitnegCD71highTer119neg stage E2 population. Towards characterizing this dynamic new cohort, we've optimized serum-free systems for murine BM erythroblast development, and have purified stage E2 erythroblasts, their stage-E1 KitposCD71highTer119neg progenitors, and stage-E3 KitnegCD71highTer119pos progeny. Upon profiling each, we discovered two E3 ubiquitin ligase adaptors to be selectively expressed at high, sustained levels in late-stage murine and human BM erythroblasts (and subject to GATA1 regulation), TRIB3 pseudokinase and SPRY1. We've further constructed conditional Trib3-/- and Spry1-/- mouse models - and in each have observed selectively skewed erythropoiesis at steady-state; and markedly compromised erythropoiesis during anemia. In Aim #1, we now propose to define specific stages, and physiological conditions, during which TRIB3 and SPRY1 regulate erythroblast development. Effects on cellular pathways also will be investigated; and co-IP plus mass spectrometry approaches will be used to identify TRIB3 and SPRY1 complexed E3 ubiquitin ligases plus coupled erythroid targets. In Aim #2, we will apply transcriptome and phosphoproteome approaches to test the hypothesis that the above stage E2 erythroblasts are dynamically advantaged in their expansion capacities via stage-specific expression of cell surface adhesion factors that mediate their selective interactions with a supporting BM stromal cell component. Studies promise to provide new insight into factors that (dys)regulate late-stage erythroblast and red cell production; and may uncover rational targets for new anti-anemia agents (as urgently needed in chemotherapy contexts)(8-10). PUBLIC HEALTH RELEVANCE: Hour-to-hour bone marrow production of red blood cells (RBC) is essential for tissue oxygenation. Faltered RBC formation results in health-compromising anemia as associated with cancer therapies, renal disease, aging, AIDS, and inherited RBC disorders (eg, sickle cell anemia). Proposed work promises to discover and characterize novel regulators of erythroblast and RBC production as potentially drugable E3 ligase complexes; and as a new RBC progenitor niche in bone marrow with unique expansion potential.